Project Summary Reactivation of pathways used during embryonic development to pattern and specify tissue fate and function can play a role in disease. This proposal explores the idea that abnormal re-activation of the developmentally important transcription factor GATA4 in the stratified epithelium of the esophagus contributes to the pathology of Barrett's esophagus (BE), a premalignant metaplasia of the esophagus. Individuals with BE have an elevated lifetime risk of developing esophageal adenocarcinoma (EAC), a deadly cancer with a five-year survival rate of only ~18%. As the most widely recognized EAC risk factor, BE is a critical target for the development of therapies that can reduce BE and thereby esophageal cancer. Our previous work demonstrates that GATA4, which is expressed in the jejunum but absent in the ileum of the small intestine, is essential to pattern the jejunal-ileal boundary. GATA4 is also differentially expressed at the squamocolumnar junction, where it is present within the columnar epithelium of the glandular stomach but absent from the stratified squamous epithelium of the esophagus/forestomach. In BE, this boundary is disrupted with the stratified squamous epithelium of the esophagus being replaced by columnar epithelium. The lack of GATA4 expression in normal esophageal epithelium and its presence in areas of BE metaplasia along with the observation that the GATA4 gene is frequently amplified and expressed in EAC suggest a role for GATA4 in BE pathogenesis. Moreover, acid and bile, two gastroesophageal reflux components implicated as having key roles in BE development, induce GATA4 expression in adult human esophageal squamous cells. We hypothesize that exclusion of GATA4 from esophageal/forestomach epithelium is essential to establish a normal squamocolumnar junction during development and that re-activation of GATA4 in stratified epithelium in the setting of reflux-induced inflammation contributes to development of Barrett's esophagus. We will test our hypotheses using unique mouse and human models to manipulate GATA4 expression and function. In Aim 1, we will determine the role of GATA4 in defining the squamocolumnar junction during development. Our preliminary data showing conversion of columnar epithelium to stratified epithelium in GATA4-deficient hindstomach and conversion of stratified epithelium to columnar epithelium in GATA4-expressing forestomach support our hypothesis. In Aim 2, we will determine how re-activation of GATA4 in stratified epithelium in the setting of reflux-induced inflammation contributes to the pathogenesis of Barrett's esophagus. We expect that studies of mice with altered GATA4 expression within the stomach and of human esophageal organoids expressing GATA4 will provide new models to study BE. These studies are significant because, if our hypothesis is correct, we will identify GATA4 and its downstream targets as possible targets to interrogate for future therapies and/or as biomarkers for BE and other epithelial metaplasias.